The rubella virion is a member of the Togavirus family, and is a spherical, enveloped virus approximately 60 nm in diameter. The virion consists of a 10 kb single-stranded RNA molecule encapsidated in an icosahedral nucleocapsid and surrounded by a lipid envelope. Multiple copies of a capsid (C) protein make up the nucleocapsid. The envelope consists of lipoproteins derived from the infected host cell and two viral glycoproteins, designated E1 (53-58 kDa) and E2 (42-48 kDa) (Waxham and Wolinsky, Virology 143:153-165, 1985).
Primary rubella infection is characterized in most individuals by the presence of a macropapular rash, fever, malaise, and lymphadenopathy. Rubella is typically a mild and self-limited disease, and is most often contracted during childhood. Primary infection in adults is less common, and may have very serious consequences in pregnant women. Infection of a fetus during the first trimester of pregnancy may result in spontaneous abortion or severe fetal abnormalities. A congenitally infected infant may exhibit one or more of a variety of birth defects collectively known as congenital rubella syndrome (CRS). Common birth defects associated with CRS include cataracts, central nervous system deficits, microcephaly, motor deficits, deafness, congenital heart disease, and mental retardation.
Because of the fetal risk associated with primary maternal rubella infection, test samples from pregnant women are routinely screened for the presence of anti-rubella IgM antibodies during the first trimester of pregnancy. Primary rubella infection is associated with a pronounced specific IgM antibody response, while reinfection (often asymptomatic) is characterized by elevated levels of specific IgG antibodies in the absence of detectable levels of specific IgM antibodies. Unlike primary infection, reinfection of immune pregnant women is generally thought to be harmless to the developing fetus. When prenatal screening indicates that a woman has acquired a primary rubella infection during the early stages of pregnancy, a therapeutic abortion is often recommended. As a result, it is imperative that the test results are accurate.
A variety of methods related to the detection of anti-rubella IgM antibodies have been described. Initial assays relied on hemagglutination inhibition (HAI) testing, which is dependent upon the hemagglutinating properties of the viral E1 and E2 glycoproteins. If a biological sample to be tested contains antibodies directed against these viral hemagglutinins, rubella virus can no longer bind to red blood cells (usually from chicken blood) and this inhibits hemagglutination (see e.g. Peetermans and Huygelen, Presse Med. 75:2177-2178, 1967). Unfortunately, titers of these anti-hemagglutinin antibodies increase significantly following both primary infection and reinfection, and so this method cannot be used to distinguish between maternal infections likely to result in fetal defects and maternal infections unlikely to affect the fetus.
More recently, enzyme-linked immunosorbent assays (ELISAs) have become the method of choice in the diagnosis of primary rubella infection (see e.g. Steece et al., J. Clin. Microbiol. 21(1):140-142, 1985). In most cases, rubella viral capsid or envelope glycoprotein antigens (whole proteins, viral extracts, or peptides) are immobilized on a solid support and exposed to a biological sample to be tested for the presence of anti-rubella antibodies. Antigens previously described include novel linear and cyclic peptides corresponding to regions of the rubella E1 and C proteins (U.S. Pat. Nos. 5,164,481 and 5,298,596), novel linear and cyclic peptides corresponding to regions of the E1 and E2 glycoproteins (U.S. Pat. No. 5,427,792) and intact rubella virus (or antigens or fragments thereof) in which oligosaccharide moieties have been modified for better recognition by antibodies (U.S. Pat. No. 4,965,069). Any anti-rubella antibodies present in the test sample bind to the immobilized antigen. After appropriate washing, the presence or absence of bound antibody is detected through the use of an indicator reagent capable of complexing with an anti-rubella IgM antibody. This indicator reagent is typically conjugated to a detectable label. After washing, the bound detectable label is quantified directly or indirectly, and the result is used to determine whether or not the initial sample contained specific anti-rubella IgM antibodies. The presence of specific anti-rubella IgM antibodies in a sample forms the basis for a diagnosis of primary rubella infection.
Unfortunately, there is a disturbing lack of specificity in existing anti-rubella IgM immunoassays. False positive results are obtained so frequently that most laboratories use a complicated testing algorithm to confirm an occurrence of primary rubella infection. This algorithm includes repeated testing of each sample employing different anti-rubella IgM diagnostic kits, a time-consuming and expensive process. The algorithm may also include complementary testing for specific anti-rubella IgG antibody avidity. The initial immune response following exposure to a novel antigen is characterized by the production of an abundance of IgM antibodies, whereas the specific high-avidity IgG antibody response is characteristic of secondary responses and reinfection. Consequently, if IgG antibodies from a patient sample taken during early pregnancy bind to rubella antigens with high avidity, it is unlikely that the initial maternal infection occurred during the critical early stages of fetal development. Conversely, if the IgG antibodies in the patient sample bind the antigen with low avidity, it is suggestive of a recent primary infection with the rubella virus.
Several potential causative factors have been implicated in the lack of specificity in anti-rubella IgM immunoassays, and attempts have previously been made to reduce the incidence of false positive assay results. For example, Macioszek et al. (U.S. Pat. No. 5,698,393, issued Dec. 16, 1997; “Macioszek”) disclose a method for reducing or eliminating false positive IgM immunoassay results caused by the presence of rheumatoid factors (autoantibodies against human IgG, usually of the IgM isotype) in tested samples. This method involves treatment of samples suspected of containing rheumatoid factors (RF), either directly or while in complex bound to the solid phase, with a RF neutralization citrate buffer of a pH most preferably between 3.5 and 5.0. Macioszek teaches that the binding of nonspecific IgM molecules such as RFs to anti-rubella IgG molecules captured by the affixed antigen is typically disrupted within this pH range, while binding of specific IgM antibodies to immobilized rubella antigen is not disrupted. This patent does not address the problem of assay nonspecificity resulting from the presence of causative factors other than RF, and RF are only present in a subset of tested sera.
Fabrizi et al. (U.S. Pat. No. 5,300,427, issued Apr. 5, 1994; “Fabrizi”) also disclose a method for reducing nonspecific signal generated in IgM ELISAs. Examples include an assay for IgM molecules recognizing “extractive antigens” of rubella. This method comprises dilution of serum with buffer containing type IV collagenase prior to placing the serum in contact with the solid support, upon which is affixed anti-IgM antibodies. The complement system is composed of a set of plasma proteins that attack extracellular pathogens. One of these complement proteins, C1q, which is capable of binding serum IgM antibodies, allegedly interacts nonspecifically with the enzyme-conjugated secondary antibody. Fabrizi teaches that collagenase aids in the separation of serum IgM molecules from attached C1q complement molecules, and thereby improves the specificity of the immunoassay by eliminating false positive signals resulting from the abovementioned nonspecific interaction. This patent does not address the problem of assay nonspecificity resulting from the presence of factors other than crossreactive C1q protein in the biological sample.
There exists a need for an improved immunoassay for the detection of anti-rubella IgM antibodies which eliminates all or most of the false positive results generated by current assays without affecting the sensitivity of the assay.